A novel missense mutation of Mip causes semi-dominant cataracts in the Nat mouse

Abstract

Major intrinsic protein of lens fiber (MIP) is one of the proteins essential for maintaining lens transparency while also contributing to dominant cataracts in humans. The Nodai cataract (Nat) mice harbor a spontaneous mutation in Mip and develop early-onset nuclear cataracts. The Nat mutation is a c.631G>A mutation (Mip^Nat), resulting in a glycine-to-arginine substitution (p.Gly211Arg) in the sixth transmembrane domain. The Mip^Nat/Nat homozygotes exhibit congenital cataracts caused by the degeneration of lens fiber cells. MIP normally localizes to the lens fiber cell membranes. However, the Mip^Nat/Nat mice were found to lack an organelle-free zone, and the MIP was mislocalized to the nuclear membrane and perinuclear region. Furthermore, the Mip^Nat/+ mice exhibited milder cataracts than Mip^Nat/Nat mice due to the slight degeneration of the lens fiber cells. Although there were no differences in the localization of MIP to the membranes of lens fiber cells in Mip^Nat/+ mice compared to that in wild-type mice, the protein levels of MIP were significantly reduced in the eyes. These findings suggest that cataractogenesis in Mip^Nat mutants are caused by defects in MIP expression. Overall, the Mip^Nat mice offer a novel model to better understand the phenotypes and mechanisms for the development of cataracts in patients that carry missense mutations in MIP.

Keywords: MIP; congenital cataract; missense mutation; mouse; semi-dominant cataract.

Fig. 1.

Lens opacities in Nat mutant mice. A–D. Gross appearances of the eyes among the wild-type (A), Nat/Nat homozygous (B) and Nat/+ heterozygous (C and D) mice at 3 weeks (A–C) and 12 months (D) of age. The magnified images in the area of eyes are shown in A’–D’. A’’–D’’’. Phenotypic comparisons via dark field microscopy of the diagonal side views of the eyes (A’’–C’’) and the anterior views of the lens among each mouse at 3 weeks of age (A’’’–C’’’) and Nat/+ heterozygous mouse at 12 months of age (D’’ and D’’’). Arrows and arrowheads indicate profound lens opacities in Nat/Nat mice and mild disorganization of the lens fibers in Nat/+ mice, respectively. Scale bar=1 mm.

Fig. 2.

Lens histology in Nat mutant mice. A comparison of the histological lens phenotypes among the wild-type (A and D), Nat/Nat (B and E), and Nat/+ mice (C and F) at P0 (A–C) and P30 (D–F). Arrows and arrowheads indicate the swelling of the lens fiber cells and small vacuoles on the lens fibers, respectively. Scale bar=100 µm.

Fig. 3.

The Nat mutation in Mip. A. Mapping of the Nat locus. The diagram shows the fine-mapping results for the genomic interval between the telomere and 118.2 Mb region on chromosome 10 that was linked to the normal and cataract phenotypes observed in [(SJL/J-Nat/Nat × BALB/cA) F₁ × SJL/J-Nat/Nat] backcrossed mice. NL: normal lens. B. Sequence analysis of the wild-type, Nat/+, and Nat/Nat mice revealing a c.631G>A substitution (arrows) in Mip, resulting in a p.Gly211Arg substitution. C. The c.631G>A introduces a BslI site, facilitating genotyping of the mice via PCR-RFLP analysis. M: size standard (100-bp ladder). D. Schematic diagram of the MIP secondary structure showing the locations of the mutations in humans (green), mice (blue), and rat (black). The six transmembrane domains (H1, H2, H3, H4, H5, and H6), two hemichannels (HB and HE), and the extracellular (LA, LC, and LE) and intracellular (LB and LD) loops are indicated.

Fig. 4.

Evolutionary and functional impact of the Nat mutation (p.Gly211Arg) in MIP. A. Evolutionary conservation of Gly211 in MIP. The top diagram shows an interspecies alignment. Arrow indicates the p.Gly211Arg mutation site. The bottom diagram indicates the conservation rates among the seven species. B. Ribbon diagram representations of MIP (PBD: 2B6O) [11]. The Gly211 residue in the H6 domain is shown as a colored atom sphere. C. Stick representation of residue 211 of MIP on a B-spline diagram showing the substitution from glycine (neutral, left) to arginine (positive charged, right). D. Space-filling model of Gly211 (left) or Arg211 (right).

Fig. 5.

Quantitative analyses of Mip transcript and MIP protein. A. Relative levels of Mip mRNA in the eye of wild-type, Mip^Nat/+, and Mip^Nat/Nat mice at P0. B. Western blot analysis of MIP protein derived from the eye of wild-type, Mip^Nat/+, and Mip^Nat/Nat mice at P1. Note the stepwise reduction of a single band at approximately 26-kDa as recognized by an anti-MIP antibody. The samples were processed for indirect immunofluorescence using an anti-CTNNB1 antibody. M: size standard (protein ladder). C. Relative levels of MIP proteins as detected by western blot analysis. **P<0.01.

Fig. 6.

Immunohistochemistry of MIP in the lens of Nat mutants. A. Immunofluorescent staining with anti-MIP antibody (green) and DAPI (blue) showing normal expression and localization in the whole lens (left), the equator region (Eq) (middle), and the organelle-free zone (OFZ) (right) of adult wild-type mice at P70. As, anterior segment; Ps, posterior segment; and Le, lens epithelium. B–M. Immunofluorescent staining for MIP (green), CTNNB1 (red), and DAPI (blue) in the lens of wild-type (B, C, H, and I), Mip^Nat/Nat (D, E, J, and K), and Mip^Nat/+ (F, G, L, and M) mice at P0 (B–G) and P30 (H–M). Highly magnified images of the Eq (B, D, F, H, J, and L) and OFZ (C, E, G, I, K, and M) are shown. Scale bar=100 µm.